One of the most important problems yet to be solved in biochemistry concerns the details of how the cell regulates its own biosynthesis. The understanding of cellular regulation would have great impact on our understanding of cellular differentiation and as a consequence of this, cures for cancer and birth defects may be found. The long term goal of this research is to examine the molecular details of the function of the protein molecules involved in the regulatory process. Emphasis will be directed towards the pyrimidine biosynthesis pathway, the products of which are necessary for DNA replication. Of particular concern to us, is the enzyme aspartate transcarbamylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms. In order to produce detailed information concerning how this enzyme functions on the molecular level, we have isolated a series of mutant aspartate transcarbamylase molecules with single amino acid substitutions. The production of these mutant enzymes was achieved by suppression of a nonsense codon situated in the gene coding for the enzyme. These mutants are particularly important because they still retain enzymatic activity and many possess altered regulatory functions. Now that characterization of enzymes in crude form is complete, the most significant mutant enzymes will be purified to homogeneity and subjected to a comprehensive analysis in order to determine exactly what alterations the substitution has created. Correlations will be made between the altered biochemical properties of the mutants and the perturbations of the three-dimensional structure of th enzyme caused by the amino acid substitution. Compilation of data for a number of these mutants should allow us to propose a molecular mechanism by which aspartate transcarbamylase exerts allosteric control over the pyrimidine pathway. By use of both a mutator gene and reversions, additional single amino acid substitution mutants will be added to our collection. Further studies are planned both of the wild-type enzyme, and of the other control mechanisms government the pyrimidine pathway.